There is a significant clinical need for novel methods of detection and treatment of gene disorders and diseases, such as cancer, that offer improved sensitivity, specificity, and cost-effectiveness. The object of any gene or drug therapy is to safely deliver therapeutic agents inside the cell. It has been more than 12 years since the first gene therapy trial, and to date, after much intense research and more than 600 clinical trials, no gene therapy has been approved. The main hurdle to overcome in this field is the lack of efficient, specific and safe nucleic acid (DNA, miRNA or sRNA) delivery systems. For successful delivery, the therapeutic agent or imaging agent/nucleic acid should be delivered to the target tissue or cell type. Once delivered to the local target site, the agent should be able to enter the cell for imaging or repair. The delivery device should be smaller than the size of the target cell in order to achieve entry into the cell. Chan et al., Nano Lett.; 6(4), 662-668 (2006) have reported the effect of the size of a delivery device on cell penetration. According to the research, a device which is nano-meters in size could enter a cell whose size is higher than micrometers.
In recent years, polymeric micelles have been the object of growing scientific attention. Polymeric micelles have emerged as a potential carrier for biomolecules for several reasons. They can solubilise biomolecules in their inner core, they offer attractive characteristics in spatial dimensions (>100 nm) for cell entry and they have the capacity to evade scavenging by the mononuclear phagocyte system. Advantageously, micelle forming polymers usually contain block co-polymers, which are found in sequences of hydrophobic blocks of the copolymer comprised of poly(caprolactone), poly(d,l-lactide) or poly(propylene) with a hydrophilic block of poly(ethylene glycol) PEG segments. With such polymers, it is possible to design a system that does not precipitate out of solution, is stable and contains a large number of distinct microscopic domains. These domains usually possess hydrophobic cores and highly hydrated hydrophilic shells or coronas. Current micellar systems however are only responsive to a limited extent to their biological environment and cannot be functionalized for specific delivery to a target cell.
Recently, nanoparticles are thought to have potential as novel probes for both diagnostic (e.g. imaging) and therapeutic purposes (e.g. drug delivery). In particular, specialized nanoparticle systems known as nanoshells, have shown promise in delivering genes to cells. However, these nanoshells are generally made of non-biodegradable materials, for example, synthetic polymers such as polyallylamine hydrochloride or inorganic material such as gold or silica, which have long-term biocompatibility concerns.
A number of other techniques have been investigated to direct therapeutics and diagnostic agents to tumours. These have included targeting of tumour cell surface molecules, targeting regions of activated endothelium, utilizing the dense and leaky vasculature associated with tumours and taking advantage of the enhanced metabolic and proteolytic activities associated with tumours. Antibody labelling has been used to achieve cell-selective targeting of therapeutic and diagnostic agents. A number of approaches have been taken for antibody-targeting of therapeutic agents. These have included direct conjugation of antibodies to drugs such as interferon alpha, tumour necrosis factor and saporin. Antibody conjugation has also been used for tumour-targeting of radioisotopes for radioimmunotherapy and radioimmunodetection. Currently, there is a commercial product for detection of prostate cancer (ProstaScint) that is an antibody against prostate-specific membrane antigen conjugated to a scintigraphic target.
International Patent Publication No. WO 01/64164 describes labelled nanocapsules comprising DNA within surfactant micelles and encapsulated within a biocompatible hydrophilic polymer.
Virus particles have been developed that display single chain antibodies on their surface, allowing specific targeting of a wide variety of cell types. In order to target regions of activated endothelium, immunoliposomes have been made with antibodies to E-selectin on their surfaces. Recently tumours have been imaged using protease-activated near infrared fluorescent probes.
Over the past several years, there has been increasing interest in combining emerging optical technologies with the development of novel exogenous contrast agents, designed to probe the molecular specific signatures of cancer and to improve the detection limits and the clinical effectiveness of optical imaging.
Sokolov et al (Cancer Research 63, 1999-2004 (2003)) recently demonstrated the use of gold colloid conjugated to antibodies to the epidermal growth factor receptor (EGFR) as scattering contrast agents for biomolecular optical imaging of cervical cancer cells and tissue specimens. In addition, multiple groups including Bruchez et al. (Science 281, 2013-2016 (1998)) and Akerman et al. (Proc. Natl. Acad. Sci. U.S.A., 99, 12617-12621 (2002)) have disclosed optical imaging applications of nanocrystal bioconjugates. More recently, interest has developed in the creation of nanotechnology-based platform technologies, which couple molecular specific early detection strategies with appropriate therapeutic intervention and monitoring capabilities.
Nedeljkovic and Patel (Appl. Phys. Lett., 58, 2461-63, (1991)) disclosed silver-coated silver bromide particles that are produced by intense UV irradiation of a mixture of silver bromide, silver, sodium dodecylsulfate (SDS) and ethylenediaminetetraacetic acid (EDTA). The Neideljkovic particles range in size from approximately 10 to 40 nm and are irregularly shaped as determined by transmission electron microscopy. Predictably, the spectra obtained from these particle preparations are extremely broad.
U.S. Pat. No. 5,023,139 discloses theoretical calculations indicating that metal-coated, semiconducting, nanometer-sized particles should exhibit third-order non-linear optical susceptibility relative to uncoated dielectric nanoparticles. This is due to local field enhancement. In those embodiments that do in fact propose a metal outer shell, there is an additional requirement as to the specific medium in which they must be used in order to properly function. Shell sizes have, in general, been relatively large, usually of the order of about 5 μm. In drug delivery applications, a smaller particle diameter is important for prolonged blood circulation and enhanced drug targeting to specific body sites.
U.S. Pat. No. 6,479,146 describes a process for preparing coated particles and hollow shells by coating colloidal particles with alternating layers of oppositely charged nanoparticles and polyelectrolytes, and optionally removing the colloidal cores. The process involves the preparation of hollow silica microspheres via layer-by-layer shell assembly on 640 nm diameter polystyrene latex particles, followed by pyrolysis at 500° C. to decompose the polystyrene core. The same assembly procedure was also used to prepare silica-containing shells on 3 μm diameter melamine-formaldehyde particles, followed by acid dissolution of the core.
International Publication No. WO 2005/044224 describes a drug delivery system based on polymer nanoshells. In certain embodiments, the polymeric nanoshells comprise one or more polymeric shells around a hollow core. In other embodiments, nanoshells are described which are useful for the delivery of agents such as, for example, various diagnostic and therapeutic agents. The nanoshells disclosed are preferably composed of biocompatible organic polymers which are most preferably biodegradable as well. The shell layers can comprise materials such as gelatin, chitosan, dextrate sulphate, carboxymethyl cellulose, sodium alginate, poly(styrene sulfonate) (PSS), poly(lysine), poly(acrylic acid), poly(dimethyldiallyl ammonium chloride) (PDDA) and poly (allylamine hydrochloride) (PAH). However, the shells described are composed of an electrostatic interaction multilayer-based membrane. Thus negative and positive charges are required on the particle, the electrostatic nature of the surface induces interactions with proteins and lipoproteins during the blood circulation. Such non-specific interaction can decrease the nanoparticle lifetime.
Hu et al (Polymer, Vol. 46, Issue 26, 2005 pg. 12703-12710) describe formation of hollow polymeric nanospheres based on a core-template-free route, and the effects of polymerization concentration, shell cross-linking, pH, salt concentration and temperature on the size and stability of hollow polymeric nanospheres. The hollow structure of polymeric nanospheres is spontaneously formed by polymerization of acrylic acid monomers inside the chitosan-acrylic acid assemblies. The size of the hollow nanospheres can be manipulated by changing pH, salt concentration and temperature.
Li et al (Colloid & Polymer Science, Vol. 286, 6-7, pg. 819-825) describe biodegradable chitosan hollow microspheres where are prepared using uniform sulfonated polystyrene (PS) particles as templates. The chitosan was adsorbed onto the surface of the sulfonated polystyrene templates through the electrostatic interaction between the sulfonic acid groups on the templates and the amino groups on the chitosan and crosslinked using glutaraldehyde. The controlled release behavior of the chitosan hollow microspheres was also primarily investigated after template removal.
The limited success of current pharmaceutical therapies is due to the absence of an innovative drug delivery system which can increase the safety and efficacy levels but also improve the overall performance of the therapeutic molecule. Stability and degradability control are two critical aspects that must be developed to facilitate delivery. Controlling these parameters simultaneously is a greater challenge, and most of the current vectors such as liposomes, microparticles and microemulsions do not support this, thus limiting their applications. Moreover, another major disadvantage of synthetic vectors is their low in vivo efficiency. This is a consequence of their poor targeting ability and their short lifetime due to the presence of surface positive charge or the inherently low stability of their shells (liposomes). These factors lead to the degradation of the supramolecular structure and removal by macrophages before the vector arrives at the target cell. To circumvent these problems, hollow spheres appear to be a promising strategy. Recent interest in hollow spherical structures can be attributed to their unusual properties (chemical, mechanical and optical) which suggest a wide range of applications. These structures have potential utility in encapsulation and controlled release of various biomolecules such as genes, peptides and drugs in clinical applications. Control of the structural characteristics of the hollow carrier such as shell thickness, surface charge, pore size and mechanical strength is essential to achieving the aim of realising an ideal encapsulation system.
Clearly, there is a need therefore to develop the next generation of hollow nanospheres that are more robust, biocompatible and are responsive to their environment, thus triggering the smart release of the biomolecule after delivery to the target. It is desirable that these polymeric systems would be biocompatible, capable of carrying a high payload, capable of acting as a reservoir and could be programmed to respond to temperature and the like and be modified to target drug delivery to a specific site.
It is thus an object of the present invention to provide improved biodegradable hollow mono-dispersed nanospheres which are adaptable to target specific sites for use as carriers of and to allow targeted and controlled delivery of biomolecules and therapeutic agents and the like or for use in other nanosphere applications.
It is a further object of the invention to provide biodegradable nanoshells which are adaptable to target specific sites, thereby allowing targeted and controlled delivery of biomolecules and therapeutic agents and the like.